1. Field of the Invention
This invention relates to a platelet aggregation reagent for EDTA-anticoagulated blood, a reagent container and a method of measuring platelet aggregation using a standard platelet counter which is cost-effective, simple, overcomes storage problems and can be applied for routine analysis utilizing very small samples.
2. Description of the Background
Platelets play an important role in coagulation. Although their ability to function properly is affected by many common drugs such as aspirin, the proper functioning of platelets is not routinely tested because no cost-effective tests exist at the present time.
When an injury occurs, platelets adhere to the damaged tissue and a platelet aggregate or platelet plug is then formed. Coagulation is initiated thereafter, ultimately leading to fibrin formation which strengthens the platelet plug.
The activation of platelets and their response play a key role in hemostasis. At present, the number of platelets in the blood is routinely measured by means of hematology counting instruments. Although the number of platelets varies considerably from person to person, these variations are of little importance as long as platelet function is preserved. For example, a platelet count of 150,000-400,000/mm.sup.3 is normal but adequate platelet function can be observed even at counts as low as 50,000/mm.sup.3.
Platelet counts, which are done routinely, are probably of less importance than platelet function which is rarely done. However, in practice, many more platelet counts than platelet function tests are performed . This is most likely due to the development of routine systems for counting platelets whereas methods for measuring platelet function are tedious and expensive. By means of example, a platelet function test may cost thirty times more than a platelet count (about $5.00 vs. $150.00).
There are many instances where testing platelet function would be of use. These are cases of either hereditary conditions in which there is a genetic absence of some part of the system, or situations where the interference occurs in the steps leading to activation of the platelets.
Examples of hereditary platelet disorders are afibrinogenemia, Bernard-Soulier syndrome, connective tissue abnormalities, Glanzmann's thrombasthenia, glycogen storage disease, macrothrombocytothia, nephritis and deafness, homocystinuria, May-Hegglin anomaly, dystrophia myotonica, storage pool defects including Chediak-Higashi syndrome, thrombocytopenia with absent radi, Wiskott-Aldrich syndrome and Hermansky-Pedlak syndrome, Wilson's disease, and swiss cheese platelets, among others.
In addition, there also exist acquired platelet disorders. Examples of acquired platelet disorders are aspirin ingestion, asthma, hypothyroidism, ethanol-induced, hypercoagulability, immune thrombocytopenia, lupus erythematosus, myeloproliferative disorders including myeloid metaplasia polycythemia rubra vera, scurvy, idiopathic thrombocytopenia purpura, sideroblastic anemia, thrombocythemia, Von Willebrand's syndrome, uremia and abnormal release mechanism (aspirin-like defect), among others.
Most notable among the acquired disorders is that resulting from ingestion of aspirin. In addition there is a large number of other drugs that interfere with platelet function However, none is more ubiquitous than aspirin.
Various methods have been known for measuring platelet function. Among them, is aggregometry which is described by Born, G. B. R., in "Quantitated Investigations into the Aggregation of Blood Platelets", J. Physiol.162:67-68(1962).
Aggregometry is a turbidometric method for measuring platelet aggregation. Platelet-rich plasma is prepared from whole blood by centrifugation to remove the red blood cells. The platelets are placed in a cuvette at 37.degree. C. with a stirrer, and a platelet activator such as collagen is added to induce platelet aggregation. The aggregation of the platelets reduces the turbidity of the platelet suspension and more light passes through the cuvette. Changes in transmitted light are recorded as a voltage change on a chart on a chart recorder.
Cardinal D. C., and Flower, R. J., in "The Electronic Aggregometer: a Novel Device for Assessing Platelet Behavior in Blood", J. Pharmacol. Methods 3:135-158(1980) describe a method of measuring aggregation by impedance. This is a relatively new method utilizing whole blood. In this method, aggregation is measured as changes in impedance which occur as platelets aggregate on platinum electrodes.
Methods using platelet counters are the most aoourate for measuring platelet aggregation since only single platelets are counted (perhaps doublets).
Malinski, J. A., and Nelsestuen, G. L., in "Relationship of Turbidity to the Stages of Platelet Aggregation", Biochim. et Biophys. Acta 882:177-182(1986), have shown that small aggregates of platelets give increased instead of decreased turbidity in an aggregometer. In this method, very large aggregates are required to produce a decrease in light scattering. Therefore, turbidity can increase or decrease with platelet aggregation depending on the size of the aggregates. Thus, it is argued in the reference, aggregometry is subject to variations that result from the size of the aggregates. Platelet counters, on the contrary, are not subject to these interpretive difficulties.
The use of platelet counters to measure platelet aggregation has been discussed by several authors (Higashi, T., Hashimoto, M., Kakishita, E., and Nagai, K., in "The Changes in the Number of Single Platelets in Human and Rabbit Platelet Aggregation Measured by Electric Particle Counting Method", Thrombosis Res. 21:457-468(1981); Butchers, J., Humphrey P. P. A., Hyde, J. J., Lumley, P., and Spurling, N. W., in "The Evaluation of a New Electronic Counting Technique for Measurement of Platelet Aggregation in Human Whole Blood in vitro", British J. Pharmacol. 70:160-161(1980); Saniabadi, A. R., Lowe, G. D. O., Forbes, C. D., Prentice, C. R. M., and Barbenel, J. C., in "Platelet Aggregation Studies in Whole Human Blood," Thrombosis Res. 30:625-632(1983); Lumley, P., and Humphrey, P. A., in "A Method for Quantitating Platelet Aggregation and Analyzing Drug-receptor Interactions on Platelets in Whole Blood in vitro", J. Pharmacol. Methods 6:153-166(1981)).
The method used by these investigators consists of warming blood or plasma in a water bath at 37.degree. C. and stirring with a magnetic stirrer. Blood for this procedure is collected in sodium citrate which causes spontaneous platelet aggregation. It has been reported that in as short a period as 40 minutes, 40% of the platelets may be lost (Fox, S. C., Burgess-Wilson, M., Heptinstall, S., and Mitchell, J. R. A., in "Platelet Aggregation in Whole Blood Determined Using the Ultra-Flo 100 Platelet Counter", Thromb. Haemostas. (Stuttgart) 48:327-329(1982)). This results in problems of storage and collection.
Accordingly, there is still a need for an improved method of testing platelet function which is reliable, cost-effective and easy to implement with readily available instrumentation while at the same time overcoming storage problems encountered by prior art procedures.